Novel methods and compositions are provided for the biocontrol of plant diseases by use of a biological coating that has both a protective and a curative effect for the control of postharvest decay. The coating includes chitosan salts, at least one antagonistic microorganism, and a softener.
The U.S. market for biocontrol of tree fruit postharvest diseases could exceed $100 million by the year 2000 (Industrial Bioprocessing, September 1992). In Postharvest News and Information (1991) it was estimated that approximately 25% of harvested fruit and vegetables are lost because of postharvest diseases. Synthetic fungicides have been the primary means for controlling postharvest diseases of fruit and vegetables. However, increased concern of the public over the carcinogenicity of synthetic fungicides, has led to the withdrawal of some fungicides from the market. The development of fungicide-resistance in pathogens has limited chemical fungicides as a means of controlling them.
Control of plant diseases is not a problem confined to the U.S. The European Parliament has voted in favor of a total ban on postharvest treatment of fruits and vegetables with pesticides as soon as this ban becomes feasible. The withdrawal of current fungicides from use in the United States and other parts of the world is creating a large, new market for biological control agents (xe2x80x9cbiocontrolxe2x80x9d). Baker (1987) has defined biological control as xe2x80x9cthe decrease of inoculum or the disease-producing activity of a pathogen accomplished through one or more organisms, including the host plant but excluding man.xe2x80x9d The cost of commercializing a biological control agent is much less expensive than the cost of commercializing a synthetic pesticide because only Tier 1 toxicology tests (Hofstein et al., 1994) are required. Also, if a biological control agent is properly selected, fewer new environmental impact studies are required.
Present coatings (mostly waxes) for postharvest commodities are somewhat effective in delaying ripening, but in general, do not prevent decay. Moreover, these coatings are under scrutiny as possible health hazards. Synthetic fungicides which have been added to the coatings to alleviate the problems of decay have recently been withdrawn from the market, and there is also public pressure to remove the petroleum-based coatings because of health and environmental concerns. A critical need therefore exists for alternatives to presently available coatings for agricultural commodities. The coatings need to be fungicidal as well as safe for the consumer and the environment.
Antagonistic yeasts have been reported to be effective agents for the biological control of postharvest diseases (Wilson and El Ghaouth, 1993). However, antagonistic microorganisms currently available have not been demonstrated to provide control of fruit and vegetable postharvest decay comparable to that obtained with synthetic fungicides. Limitations include the microorganisms"" inability to cure previously-established infections in the crops and to prevent the resumption of quiescent infections.
Recently, chitosan, an animal-derived polymer, has been shown to have some potential as an antifungal preservative. Chitosan, a xcex2-1,4-glucosamine polymer, is commercially produced from chitin of arthropod exoskeletons that have been deacetylated to provide sufficient free amino groups to render the polymer readily soluble in diluted organic acids. Chitosan and its derivatives are known to form a semi-permeable film (Averbach, 1978), to be inhibitory to a number of pathogenic fungi (Allan and Hadwiger, 1979), and to activate a number of biological processes in plant tissues, including the stimulation of chitinases, the accumulation of phytoalexins, the synthesis of proteinase inhibitors, and increased lignification (El Ghaouth et al., 1992, a, b). The polycationic nature of chitosan is believed to provide the basis for its physico-chemical and biological functionality. Chitosan is regarded safe as indicated by feeding trials with domestic animals. When applied as a coating, chitosan controlled decay and delayed ripening of strawberry, bell pepper, tomato, and cucumber by acting as a selective barrier to gas diffusion (El Ghaouth et al., 1991). The control of decay by chitosan is believed to originate, in part, from its antifungal property. Indeed, in vitro studies showed that chitosan not only inhibited the radical growth of major postharvest pathogens, but also induced severe morphological alterations in Rhizopus stolonifer and Botrytis cinerea, as well as increased cellular leakage in both fungi, presumably by interfering with fungal plasma membranes (El Ghaouth et al., 1992 b).
Because infection of commodities such as fruit can occur either prior to harvest or during harvesting, an ideal biological control agent is expected to display both a protective and curative activity comparable to that observed with synthetic fungicides. Currently available antagonistic microorganisms do not appear to be able to control previously-established infections and are most effective when applied prior to infection by the pathogen.
New safe and effective means of controlling postharvest diseases are needed. The present invention provides such means.
The present invention is directed to unique compositions that are a combination of antifungal agents with antagonistic microorganisms and a softener. The invention also relates to methods of preventing and curing postharvest decay of plants caused by various pathogenic fungi, by applying the compositions of the invention to plants.
The combinations of the present invention form a xe2x80x9cbiological coating.xe2x80x9d In an embodiment of a composition of the present invention, the antagonistic microorganism is a yeast in particular Candida saitoana and the antifungal agents include chitosan salts amended with CaCl2. Other suitable microorganisms include bacteria, for example Pseudomonas syringae and Bacillus subtilis. In a preferred embodiment, the biochemical additive are chitosan salts amended with CaCl2 or chitosan propionate amended with CaCl2, and a yeast, in particular Candida saitoana. Other suitable genera of yeast include Candida spp; Cryptococcus spp; Pichia spp; Debaryomyces spp; Bulleromyces spp; Sporobolomyces spp; and Rhodotorula spp.
Other suitable chitosan salts include chitosan acetate; chitosan sorbate; chitosan propionate; chitosan lactate; chitosan glutamate; chitosan benzoate.
A softener is an additive that renders the yeast resistant to the adverse effects of chitosan and organic acids. Monovalent and divalent cations of Ca, Mg, Zn, or K are suitable. The softener is not considered to affect the pathogen.
The combination of the antifungal property of chitosan salts e.g. chitosan acetate and the biocontrol activity of antagonistic microorganisms such as the yeast C. saitoana, wherein the yeast functions against the pathogen in the presence of chitosan salts amended with CaCl2, provides improved consistency and efficacy in controlling postharvest decay. In addition, the combination of antagonistic yeast (C. saitoana) with chitosan salts amended CaCl2 offers control of postharvest decay of fruit and vegetables superior to that obtained with antagonistic yeast alone or chitosan salts amended with CaCl2 alone. This improvement is unexpected and synergism was demonstrated.
Native chitosan and organic acids (acetic; sorbic; propionic; and lactic) are known to inhibit the growth of yeasts such as Candida spp and filamentous fungi such as Botrytis cinerea, Penicillium expansum Link, and Penicillium digitatum. Thus it is expected that the combination of chitosan-organic acid with C. saitoana will adversely affect the biocontrol activity of the selected yeast. However, unexpectedly, the combination produced improved control of pathogenic fungi.
The combination of antagonistic yeast (C. saitoana) with chitosan salts (chitosan acetate) amended with CaCl2 may be applied to harvested crops either before or after infection because the combination has both a protective and curative effect against major postharvest pathogens and offers a level of control of decay better than that of synthetic fungicides.
The complexity of the mode of action displayed by the combined agents of the present invention makes the development of pathogen resistance in the target plants more difficult and presents a highly complex disease deterrent barrier.
An aspect of the invention is a biological coating for agricultural commodities that includes, in amounts effective for biocontrol activity, chitosan salts, and at least one antagonistic microorganism effective for the biocontrol of postharvest diseases, and a softener that is, a monovalent or divalent cation, e.g. CaCl2. The effective amount of chitosan salts is from about 10 xcexcg/ml to about 500 xcexcg/ml, preferably about 20 xcexcg/ml to about 250 xcexcg/ml, more preferably about 200 xcexcg/ml. The effective amount of one antagonistic microorganism is about 107 colony forming units to about 108 colony forming units; preferably about 108 colony forming units. Another aspect of the invention is a method of protecting a commodity against postharvest decay, which includes coating the surface of the commodity by spraying, dipping, or drenching with an effective amount of the biological coating.
Fruit and plants that are targets for the methods and compositions of the present invention include pome fruit (e.g., apple, pear); stone fruit (e.g. peach, nectarine, prune); citrus fruit (e.g., orange, lemon, grapefruit, tangerine); vegetables (e.g. tomato, bell pepper, cucumber); root crops (e.g., potato, carrots); tropical fruit (e.g., mango, banana, guava, pineapple, avocado); melon fruit.
The present invention provides a novel combination of chitosan salts with at least one microorganism that is antagonistic to postharvest pathogens, and a softener. Native chitosan is known to adversely affect the viability of both antagonistic yeasts and postharvest pathogens. This previous knowledge hindered the use of chitosan salts as a potential additive to biocontrol agents (such as antagonistic yeasts). However, the addition of divalent or monovalent cations (a xe2x80x9csoftenerxe2x80x9d) such as CaCl2 to the combination of chitosan-salts with Candida saitoana allowed the antifungal property of chitosan to be exploited and the biocontrol activity of the antagonistic yeast (C. saitoana) to be enhanced. Chitosan-salt formulation that acts as a carrier for an antagonistic microorganism, with a coating that delays ripening, is inhibitory to postharvest pathogens. For example, the addition of a softener such as CaCl2 to the combination of chitosan-salts with C. saitoana allowed the antifungal property of chitosan and the biocontrol activity of C. saitoana to be exploited.
Chitosan salts; acetate; sorbate; propionate; lactate; and glutamate amended with CaCl2 are inhibitory to postharvest pathogens such as B. cinerea and P. expansum but show no effect on the growth of C. saitoana (Table 1). This was further confirmed by the results obtained with apple fruit. Apple wounds treated with a combination of C. saitoana with chitosan salts amended with CaCl2 showed an intense colonization of the wound by C. saitoana (Table 2). The complexities of this combination provide a unique composition (product). While yeasts which have been found effective include Candida saitoana (C. saitoana), Candida oleophila (C. oleophila), Candida sake (C. sake), Candida tenius (C. tenius), Candida utilis (C. utilis), Pichia guilliermondii (P. guilliermondii), it is well within the level of skill in the art to determine if a particular antagonistic microorganism shows the necessary resistance by combining the candidate microorganism with chitosan salts amended with CaCl2 in culture and observing whether or not the microorganism remains viable and grows. The combination of chitosan salt-CaCl2 with an antagonistic yeast (C. saitoana) offers effective control of a major postharvest rot of fruit and gives superior control over use of an antagonist or chitosan salts-CaCl2 alone.
The combination of chitosan-acetate-CaCl2 with C. saitoana was more effective in controlling decay of apple, orange, and lemon fruit (Table 3 and 4) than either the antagonist microorganism or chitosan-acetate-CaCl2 alone. After 14 days of storage, 23% of the lemons treated with the combination of C. saitoana with chitosan-acetate-CaCl2 and inoculated with P. digitatum were diseased (Table 4), while 75% and 35% of the fruit treated with 0.1% chitosan-acetate-CaCl2 or C. saitoana and inoculated with P. digitatum were diseased. All of the control apples inoculated with P. digitatum alone were diseased. The same pattern of decay control by the combination was also observed in apple and orange fruit (Table 3 and 4). The beneficial effect of CaCl2 is illustrated by the poor level of disease control obtained with the combination of C. saitoana with chitosan-acetate with CaCl2 (Table 3).
In addition to preventing decay, the combination of C. saitoana with chitosan-acetate-CaCl2 also displayed a curative activity against major pathogens of apple and citrus fruit (Table 5 and 6). In citrus fruit, the combination of C. saitoana with chitosan-acetate-CaCl2 was very effective in controlling early infections caused by P. digitatum. The level of control observed with the combination of C. saitoana with chitosan-acetate-CaCl2 was significantly higher than that obtained with either the antagonist or chitosan-acetate-CaCl2 alone (Table 5). Similar control of established infection by the combination of chitosan-acetate-CaCl2 was also observed with apple fruit inoculated with P. expansum (Table 6). The results from pilot trials showed that the combination of C. saitoana with chitosan-acetate-CaCl2 also was effective in controlling decay of oranges. The level of disease control obtained with the combination was comparable to that obtained with the recommended fungicide Thiabendazole (Table 7).
The curative effect of the combination is a substantial improvement over the combination of C. saitoana with glycolchitosan or carboxymethylchitosan (U.S. Pat. No. 5,633,025). The combination of C. saitoana with glycolchitosan or carboxymethylchitosan provides only protection and has no effect on established infections that occur before treatment. Because infection of fruit can occur either prior to harvest or during harvesting, an ideal biological product is expected to display both a protective and curative activity comparable to that observed with synthetic fungicides. Currently available antagonistic microorganisms do not appear to be able to control previously-established infections and are most effective when applied prior to infection by the pathogen. The lack of curative activity has been identified as a major limitation of biological approaches. The combination of C. saitoana with chitosan salts in presence of CaCl2 offers both a curative and protective activity against rot.